Ligand screening and discovery
View Patent ↗Disclosed is a method that includes: (i) providing a plurality of initial nucleic acid cassettes that include: a) a first coding region encoding a first immunoglobulin variable domain, b) a second coding region encoding a second immunoglobulin variable domain, and c) a ribosomal binding site disposed between the first and second coding regions for translation of the second polypeptide in a first expression system, wherein the first and second coding regions are in the same translational orientation; (ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the first and second region remain physically attached during the modifying; (iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells; and (iv) expressing each modified nucleic acid cassette in the transfected cells.
1. A method comprising:
(i) providing a plurality of initial nucleic acid cassettes comprising
a) a first coding region encoding a first immunoglobulin variable domain,
b) a second coding region encoding a second immunoglobulin variable domain, and
c) a ribosomal binding site disposed between the first and second coding regions for translation of the second coding region in a first expression system, wherein the first and second coding regions are in the same translational orientation and can be transcribed as a single transcript;
(ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the modifying comprises inserting a segment that comprises an internal ribosome entry site between the first and second coding regions and the first and second region remain physically attached during the modifying;
(iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells; and
(iv) expressing each modified nucleic acid cassette in the transfected cells.
2. The method of claim 1 wherein the segment further comprises a signal sequence functional in a mammalian cell.
3. The method of claim 2 wherein the segment further comprises a polyA addition regulatory sequence.
4. A method comprising:
(i) providing a plurality of initial nucleic acid cassettes comprising
a) a first coding region encoding a first immunoglobulin variable domain,
b) a second coding region encoding a second immunoglobulin variable domain, and
c) a ribosomal binding site disposed between the first and second coding regions for translation of the second coding region in a first expression system, wherein the first and second coding regions are in the same translational orientation and can be transcribed as a single transcript;
(ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the modifying comprises attaching the second coding region in frame to a sequence encoding a constant domain which comprises an Fc domain to form a fusion protein and the first and second region remain physically attached during the modifying;
(iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells; and
(iv) expressing each modified nucleic acid cassette in the transfected cells.
5. A method comprising:
(i) providing a plurality of initial nucleic acid cassettes comprising
a) a first coding region encoding a first immunoglobulin variable domain,
b) a second coding region encoding a second immunoglobulin variable domain, and
c) a ribosomal binding site disposed between the first and second coding regions for translation of the second coding region in a first expression system, wherein the first and second coding regions are in the same translational orientation and can be transcribed as a single transcript;
(ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the modifying comprises attaching the second coding region in frame to a sequence encoding a constant domain which comprises a cytotoxic domain to form a fusion protein and the first and second region remain physically attached during the modifying;
(iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells; and
(iv) expressing each modified nucleic acid cassette in the transfected cells.
6. The method of claim 5 further comprising detecting cytotoxic activity.
7. A method comprising:
(i) providing a plurality of initial nucleic acid cassettes comprising
a) a first coding region encoding a first immunoglobulin variable domain,
b) a second coding region encoding a second immunoglobulin variable domain, and
c) a ribosomal binding site disposed between the first and second coding regions for translation of the second coding region in a first expression system, wherein the first and second coding regions are in the same translational orientation and can be transcribed as a single transcript;
(ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the first and second region remain physically attached during the modifying;
(iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells;
(iv) expressing each modified nucleic acid cassette in the transfected cells; and
(v) culturing the transfected cells in a low permeability medium.
8. The method of claim 1 or 7 wherein the modifying further comprises attaching the second coding region in frame to a sequence encoding a constant domain which comprises an Fc domain to form a fusion protein.
9. The method of claim 4 wherein the modifying further comprises inserting a segment that comprises an internal ribosome entry site between the first and second coding regions.
10. The method of claim 4 further comprising producing an antibody encoded by one of the modified nucleic acid cassettes.
11. The method of claim 1 , 4 , 5 , or 7 further comprising: screening the mixture of transfected cells using fluorescence-activated cell sorting to identify transfected cell that produces at least a threshold amount of an immunoglobulin that includes the combination of first and second immunoglobulin variable domains present in an initial cassette.
12. The method of claim 1 , 4 , 5 , or 7 wherein the modifying further comprises digesting using one or more of: ApaLI, AscI, MfeI, BstEII, NotI, XbaI, NcoI, PstI, NheI, SfiI and BssH2.
13. The method of claim 12 wherein the modifying comprises releasing a segment between the first and second coding regions using AscI and MfeI; AscI and SfiI; ApaL1 and NotI; ApaL1 and NheI; or ApaL1 and BstEII.
14. The method of claim 1 , 4 , 5 , or 7 further comprising screening the transfected cells by attaching a probe to the surface of the transfected cells, the probe having a binding domain that recognizes a constant region of the fusion proteins, and detecting the interaction between the fusion protein and the surface bound probe.
15. The method of claim 1 , 4 , or 5 further comprising culturing the transfected cells in a low permeability medium.